The definitive endoderm, one of the three primary germ layers of the early embryo gives rise to the primitive gut tube, which in turn contributes to a diverse set of tissues including the thyroid, thymus, liver, lungs and digestive tract. Signals from the definitive endoderm also play essential roles during the induction and patterning of the heart and anterior CNS. We recently identified Smad2, an effector downstream of TGFb/activin/nodal signals as an essential regulator of endoderm formation in the mouse. To dissect potentially unique and/or overlapping Smad2/3 activities, Smad2 deficient ES cells transfected with expression constructs will be tested for their ability to colonize the definitive endoderm lineage. Functional activities of novel "knock-in" alleles expressing different Smad2/3 isoforms under control of endogenous Smad2 regulatory elements will also be tested. To evaluate the roles of TGFb signaling during patterning and formation of endodermal derivatives, we will exploit conditional alleles of Smad2 and Smad4 in combination with transgenic mouse strains expressing Cre in different temporal and spatial domains of the gut lineage, including the developing liver and pancreas. The zinc finger transcriptional repressor Blimp1 has been shown to control endodermal versus mesodermal fates in the frog embryo. We will generate and analyze a loss of function mutation at the mouse Blimp1 locus to test for possibly conserved function in endoderm formation. Finally to identify target genes required for endoderm formation we perform transcriptional profiling using Smad2, Smad4, Foxh1, and Mix-1 deficient ES cells induced to differentiate into endoderm in culture. Experiments outlined in this proposal aim to enhance our knowledge of how Smad2 and other transcriptional mediators control cell fate, proliferation and differentiation of the endodermal cell lineage.